Methods and compositions for the treatment of cancer

Abstract

To provide novel compositions and methods for treating a cancer.SOLUTION: Provided is a method of treating, inhibiting, and / or preventing a cancer in a subject, the method comprising: 1) administering to the subject at least one inhibitor of induction, activity, or signaling by indoleamine 2,3-dioxygenase-1 (IDO1); and 2) administering to the subject at least one second agent, the second agent being at least one anti-vasculogenic / anti-angiogenic agent and / or a therapeutic agent which acts by imposing nutrient / oxygen starvation on a cancer cell or exploiting nutrient / oxygen starvation in a cancer cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 558,573, filed on September 14, 2017, pursuant to 35 U.S.C. § 119(e). The aforementioned application is incorporated herein by reference.

[0002] This invention relates to the field of chemotherapy. Specifically, this invention provides novel compositions and methods for the treatment of cancer. [Background technology]

[0003] Angiogenesis is crucial for tumor growth (Cao et al. (2011) Sci. Transl. Med., 3:114rv3). However, unlike the strictly controlled process of angiogenesis in normal tissues, tumor angiogenesis is characterized by excessive and uncontrolled growth of blood vessels, much like that caused by ischemia in tissues such as the retina and lungs (Carmeliet, P. (2003) Nat. Med., 9:653-660). An emerging concept in the field of angiogenesis is that normalizing the pathological tumor vascular system enhances the effectiveness of chemotherapy and radiotherapy. Therefore, there is a need for new synergistic combinations of drugs that utilize tumor pathogenic angiogenesis. [Overview of the Initiative]

[0004] According to one aspect of the present invention, a method is provided for treating, inhibiting (e.g., reducing), and / or preventing cancer in a subject. The method comprises the steps of: administering at least one inhibitor of tryptophan degradation and / or induction or activity of a downstream pathway in response to this process; and administering at least one second agent, the second agent being an anti-angiogenic agent / anti-angiogenic agent and / or therapeutic agent that acts by imposing nutrient / oxygen starvation on cancer cells or by exploiting nutrient / oxygen starvation in cancer cells. In a particular embodiment, the method comprises the administration of an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor and the second agent. The combination of administered agents acts synergistically, compared to the individual activities of the agents, to inhibit, treat, and / or prevent cancer in the subject. In a particular embodiment, the IDO1 signaling inhibitor is a small molecule inhibitor (e.g., 1-methyl-tryptophan).

[0005] According to another aspect of the present invention, compositions for treating, inhibiting, and / or preventing cancer in a subject are also provided. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1A provides images of specific proliferation of 4T1 lung metastases in mice with and without IDO1. It also provides images of lungs stained with Indo ink to visualize metastatic burden in BALB / c WT and Ido1- / - mice 5 weeks after orthotopic 4T1 mammary tumor cell transplantation. Figure 1B provides representative images of immunofluorescence staining of blood vessels with anti-Cav1 and nuclei with DAPI within 4T1 lung metastases in WT and Ido1- / - mice. Figure 1C provides a quantitative assessment of angiogenesis density characterized by anti-Cav1 positive staining within 4T1 lung metastases in WT and Ido1- / - mice (N≧3 mice). Data are graphed as mean ± SEM, and significance is determined by two-sided Student's t-test. [Figure 2]Figure 2A provides representative images of immunofluorescence staining of blood vessels with anti-Cav1 and nuclei with DAPI in metastatic regions of lung specimens prepared from wild-type (WT) mice orally administered (p.o.) vehicle or 50 mg / kg epacadostat twice daily for 3 days starting from 3.5 weeks after orthotopic 4T1 breast cancer cell transplantation. Figure 2B provides a quantitative assessment of microvessel density marked by anti-Cav1 positive staining in lung metastases of WT mice that received either vehicle or epacadostat for 3 days, along with a third positive control cohort that received a single intraperitoneal injection of 50 mg / kg cyclophosphamide at 3.5 weeks, followed by assessment 3 days after administration (N≧3 mice) after orthotopic 4T1 breast cancer cell transplantation. Data are graphed as mean ± SEM, and significance is determined by one-way analysis of variance with Dunnett's multiple comparison test. [Figure 3] Figure 3A provides representative images of immunofluorescence staining of blood vessels with anti-Cav1 and nuclei with DAPI in metastatic regions of lung specimens prepared from Ido1- / - and Ifng- / -Ido1- / - mice after orthotopic 4T1 breast cancer cell transplantation. Figure 3B provides a quantitative assessment of microvessel density marked by anti-Cav1 positive staining in lung metastases of WT, Ido1- / -, Ifng- / - and Ifng- / -Ido1- / - mice after orthotopic 4T1 breast cancer cell transplantation (N≧3 mice). Data are graphed as mean ± SEM, and significance is determined by one-way analysis of variance with Tukey's multiple comparison test. Figure 3C provides images of lung staining with India ink for visualizing metastatic burden in Ido1- / - and Ifng- / -Ido1- / - mice at 5 weeks after orthotopic 4T1 breast cancer cell transplantation. Figure 3D provides Kaplan-Meier survival curves of cohorts of WT, Ido1- / -, Ifng- / - and Ifng- / -Ido1- / - mice after orthotopic engraftment of 1×104 4T1 cells (N≧17 mice), with significance assessed by log-rank test between two groups. [Figure 4]Figure 4A provides representative images of immunofluorescence staining of blood vessels with anti-Cav1 and nuclei with DAPI within metastatic areas of lung specimens prepared from WT, Il6- / -, Ifng- / -, and Ifng- / -Il6- / - mice after orthotopic 4T1 mammary tumor cell transplantation. Figure 4B provides a quantitative assessment of angiogenesis density marked by anti-Cav1 positive staining within lung metastases in WT, Il6- / -, Ifng- / -, and Ifng- / -Il6- / - mice after orthotopic 4T1 mammary tumor cell transplantation (N≧3 mice). Data are graphed as mean ± SEM, and significance is determined by one-way ANOVA with Tukey's multiple comparison test. Figure 4C provides images of lung staining with India Ink to visualize metastatic burden in Il6- / - and Ifng- / -Il6- / - mice 5 weeks after orthotopic 4T1 mammary tumor cell transplantation. Figure 4D provides Kaplan-Meier survival curves for cohorts of WT, Il6- / -, Ifng- / -, and Ifng- / -Il6- / - mice after orthotopic engraftment of 1×10⁴ 4T1 cells (N≧9 mice), whose significance was assessed by two-group log-rank tests. [Figure 5] Figure 5A shows images of metastatic pulmonary nodules and spleens stained with immunofluorescence using anti-IDO1 and anti-CD45 antibodies. Figure 5B shows images of metastatic pulmonary nodules stained with immunofluorescence using anti-IDO1 and anti-Gr1 antibodies or anti-IDO1 and anti-CD-11b antibodies. [Figure 6] Figure 6A provides flow cytometry data of immune cells isolated from 4T1 lung metastases. Figure 6B provides fluorescence microscopy images of Gr1+CD11b+ and Gr1+CD11b- classified cell populations using anti-IDO1 and anti-CD11b antibodies. Figure 6C provides images of excised Matrigel plugs containing classified Gr1+CD11b+ or Gr1+CD11b- cells transplanted into mice, and fluorescence microscopy images of invasive angiogenesis in the excised plugs stained with anti-caveolin-1 antibody. Figure 6D provides quantification of vascular density in the excised Matrigel plugs. [Figure 7]Figure 7A provides representative images of immunofluorescence staining of blood vessels with an anti-Cav1 antibody and nuclei with DAPI in metastatic regions of lung specimens prepared from WT mice orally (p.o.) administered vehicle or 50 mg / kg epacadostat twice daily for 3 days starting from 3.5 weeks after orthotopic engraftment of 4T1 breast tumor cells. Figure 7B provides representative images of immunofluorescence staining of hypoxic regions with an antibody against Hypoxyprobe™ protein adducts and immunofluorescence staining of nuclei with DAPI in metastatic regions from the same lung specimens as in Figure 7A. [Figure 8] Figure 8A provides representative images of immunofluorescence staining of blood vessels with an anti-Cav1 antibody and nuclei with DAPI in metastatic regions of lung specimens prepared from WT mice orally (p.o.) administered vehicle or 400 mg / kg indoximod twice daily for 3 days starting from 3.5 weeks after orthotopic engraftment of 4T1 breast tumor cells. Figure 8B provides representative images of immunofluorescence staining of hypoxic regions with an antibody against Hypoxyprobe™ protein adducts and immunofluorescence staining of nuclei with DAPI in metastatic regions from the same lung specimens as in Figure 8A. DETAILED DESCRIPTION OF EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0007] Chronic inflammation provides a microenvironmental context that can promote tumor promotion through complex and dynamic interactions between the stroma and tumor, which is an area of ​​active investigation (Peek et al. (2005) Cancer Res., 65:8583-8586). In a two-stage chemocarcinogenesis experimental model in which mutagenic tumor initiation and inflammatory tumor promotion are clearly separable, IDO1 (indoleamine 2,3-dioxygenase 1) has been identified as a key component of the environment that promotes inflammatory tumors (Muller et al. (2008) Proc. Natl. Acad. Sci., 105:17073-17078). Enzymatically, IDO1 catabolizes the essential amino acid tryptophan, but it is not an enzyme that maintains normal tryptophan homeostasis; this is the role of the evolutionarily convergent liver enzyme TDO2 (tryptophan dioxygenase 2). Rather, IDO1 can be expressed along various tissues, especially mucosal surfaces, and is strongly induced by the inflammatory cytokine IFNγ (interferon-γ) (Taylor et al. (1991) FASEB J., 5:2516-2522). The conceptualization of IDO1 as a regulator of immune function was revealed by the observation that depletion of tryptophan by IDO1 can suppress the activation of cytotoxic T cells (Munn et al., 1999). The demonstration that the IDO1 pathway inhibitor 1MT (1-methyl-tryptophan) can induce T cell-dependent allogeneic rejection (Munn et al. (1998) Science 281:1191-1193) dramatically strengthened the concept of IDO1 as an agent of tolerance. Subsequent findings linking the loss of the tumor suppressor gene Bin1 to IDO1 dysregulation and tumor immune evasion (Muller et al. (2005) Nat. Med., 11:312-319) provided experimental evidence for the obvious proposition that tumors may adapt this mechanism to protect “external” fetuses and overcome tumor immune surveillance.Furthermore, while IDO1 contributes to tumor development when directly expressed within tumor cells (if immunoediting is likely functioning), its expression within non-malignant stroma has also been shown to contribute to tumor development (Munn et al. (2004) J. Clin. Invest., 114:280-290).

[0008] Because the lungs are an organ with relatively high constitutive levels of IDO1, tumor development in the lungs has been studied (Takikawa et al. (1986) J. Biol. Chem., 261:3648-3653). Genetic loss of IDO1 resulted in a significant reduction in lung tumor burden in both a transgenic mouse model of de novo lung cancer and an orthotopic graft model of metastatic breast cancer. In both cases, this was due to IDO1 - / - This was translated into a significant survival benefit in animals (Smith et al. (2012) Cancer Discov., 2:722-735).

[0009] As explained above, angiogenesis is important for tumor development (Cao et al. (2011) Sci. Transl. Med., 3(114):114rv113). Unlike strictly controlled physiological angiogenesis, cancer involves excessive and uncontrolled growth of blood vessels, much like that caused by ischemia in tissues such as the retina and lungs. Experimental models of ischemia have shown that immune cells are important for pruning the excessive vascular system and limiting angiogenesis (Ishida et al. (2003) Nat. Med., 9:781-788; Wagner et al. (2008) Am. J. Physiol. Lung Cell. Mol. Physiol., 294:L351-357). Therefore, immunity may also play an important anti-angiogenic role in tumors.

[0010] One important check for tumor angiogenesis is the inflammatory cytokine IFNγ. Rather than direct tumor cell killing, IFNγ-induced loss of tumor vascular system is CD4 + and CD8+ Both are considered major mechanisms of T cell-mediated tumor rejection (Qin et al. (2000) Immunity 12:677-686; Qin et al. (2003) Cancer Res., 63:4095-4100). The inflammatory cytokine IFNγ is a major inducer of IDO1 (Yoshida et al. (1981) Proc. Natl. Acad. Sci., 78:129-132). Therefore, IDO1 may act with a negative feedback capacity to weaken the tumor-suppressive anti-angiogenic effect of IFNγ. In particular, IDO1 can enhance the induction of the inflammatory cytokine IL6 (interleukin-6) (Smith et al. (2012) Cancer Discov., 2:722-735), which is involved as a neo-angiogenic factor in tumors (Middleton et al. (2014) Crit. Rev. Oncol. Hematol., 89:129-139; McClintock et al. (2005) J. Appl. Physiol., 99:861-866). This specification shows that IDO1 plays a crucial role in supporting angiogenesis corresponding to the integration of these two competing inflammatory cytokines, IFNγ and IL6, at the interface.

[0011] The present invention provides compositions and methods for inhibiting (e.g., mitigating, slowing, etc.), preventing, and / or treating cancer. The present invention also provides compositions and methods for inhibiting, preventing, and / or treating pathogenic angiogenesis. The method comprises the steps of administering at least one inhibitor of tryptophan degradation and / or a downstream pathway that responds to this process, and at least one antivascularizing / anti-angiogenic agent and / or therapeutic agent that acts by imposing nutrient / oxygen starvation on a target tissue (e.g., cancer cells or tumors) or by utilizing the nutrient / oxygen starvation of the target tissue ("therapeutic agent") for a subject (e.g., a subject that needs it (e.g., a subject with cancer)). In certain embodiments, the method comprises the administration of an IDO1 inhibitor.

[0012] The drug administered to the subject may be included with a single composition having at least one carrier (e.g., a pharmaceutically acceptable carrier). Alternatively, the drugs may be administered separately (e.g., in separate compositions having at least one carrier (e.g., a pharmaceutically acceptable carrier)). In certain embodiments, the drugs may be administered sequentially and / or simultaneously. For example, an IDO1 signaling inhibitor may be administered before, after, and / or concurrently with an antivascular agent / anti-angiogenic agent and / or therapeutic agent. Similarly, an antivascular agent / anti-angiogenic agent and / or therapeutic agent may be administered before, after, and / or concurrently with an IDO1 signaling inhibitor. If the drugs are not administered simultaneously, they should be administered within a sufficient time interval to allow them to act synergistically in the patient.

[0013] As described above, the method comprises the step of administering an inhibitor targeting the induction or activity of tryptophan degradation, or at least one inhibitor of a downstream pathway that responds to this process. In certain embodiments, the inhibitor is a small molecule inhibitor (e.g., a small molecule inhibitor of IDO1). In certain embodiments, the inhibitor is an inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA, etc.) or a vector encoding it. In certain embodiments, the inhibitor is an antibody or antibody fragment immunologically specific to the protein being inhibited (e.g., a neutralizing antibody; e.g., an anti-IDO1 antibody or a fragment thereof). In certain embodiments, the IDO1 inhibitor is a peptide-based vaccine targeting IDO1 (e.g., Iversen et al. (2014) Clin. Cancer Res., 20:221-32). In certain embodiments, the IDO1 inhibitor does not substantially inhibit IDO2.

[0014] Examples of small molecule IDO1 inhibitors include PCT / US2014 / 022680 (e.g., tricyclic compounds related to imidazoisoindole; compounds of formula IV), PCT / US2012 / 033245 (e.g., condensed imidazole derivatives; compounds of formula I or II), PCT / US2010 / 054289 (e.g., imidazole derivatives; compounds of formulas I-VIII), PCT / US2009 / 041609 (e.g., compounds of formulas I-VIII), PCT / US2008 / 57032 (e.g., naphthoquinone derivatives; compounds of formulas I, II, or III), PCT / US2008 / 085167 (e.g., compounds of formulas I-XLIV), PCT / US2006 / 42137 (e.g., compounds of formula I), PCT / US2006 / 017983 (e.g., compounds of formula I), PCT / US2004 / 005155 (e.g., phenyl-TH-DL-trp(3-(N-phenyl-thiohydantoin)-indole), propenyl-TH-DL-trp(3-(N-allyl-thiohydantoin)-indole) and methyl-TH-DL-trp(3-(N-methyl-thiohydantoin)-indole)), P CT / US2004 / 005154 (e.g., compounds of formula I or II), U.S. Patent No. 7,705,022 (e.g., compounds of formula I), U.S. Patent No. 8,008,281 (e.g., phenyl-TH-DL-trp(3-(N-phenyl-thiohydantoin)-indole), propenyl-TH-DL-trp(3-(N-allyl-thiohydantoin)-indole), and methyl-TH-DL-trp(3-(N-methyl-thiohydantoin)-indole)), U.S. Patent No. 7,714,139 (e.g., compounds of formula I or II), U.S. U.S. Patent Application Publication No. 20140066625 (e.g., condensed imidazole derivatives; compounds of formula I or II), U.S. Patent Application Publication No. 20130177590 (e.g., N-hydroxyamidino heterocycles; compounds of formulas I-III), U.S. Patent Application Publication No. 20140023663 (e.g., 1,2,5-oxadiazole; compounds of formula I), U.S. Patent Application Publication No. 20080146624 (e.g., amidine; compounds of formula I or II), U.S. Patent Application Publication No. 20080119491 (e.g., amidino heterocycles;These compounds (compounds of formulas I-IV) are provided in U.S. Patent Application Publication No. 20080182882 (e.g., N-hydroxyamidino heterocycles; compounds of formula I), U.S. Patent Application Publication No. 20080214546 (e.g., N-hydroxyamidino heterocycles; compounds of formula I), U.S. Patent Application Publication No. 20060258719 (e.g., compounds of formula I), Banerjee et al. (2008) Oncogene 27:2851-2857 (e.g., brassin derivatives), and Kumar et al. (2008) J. Med. Chem., 51:1706-1718 (e.g., phenyl-imidazole derivatives). In certain embodiments, the IDO1 inhibitor is a prodrug (see, for example, U.S. Patent Application Publication No. 20170022157 and U.S. Provisional Application No. 62 / 555,726). All references relating particularly to the IDO1 inhibitors provided herein are incorporated herein by reference.

[0015] In certain embodiments, the IDO1 inhibitors include epacadosat (INCB024360, Incyte; Wilmington, DE; Liu et al. (2010) Blood 115(17):3520-3530; Koblish et al. (2010) Mol. Cancer Ther., 9(2):489-498), navoximod (NLG919, GDC-0919, RG6078; NewLink Genetics / Genentech), BMS-986205 (F001287, Hunt et al., AACR 2017, Abstract 4964), and PF-06840003 (Wythes et al, SITC 2016, Abstract). 253), or beta-rapacone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, Flick et al. (2013) Int. J. Tryp. Res. 6:35-45).

[0016] In certain embodiments, the IDO1 induction inhibitor is ethyl pyruvate (Muller, et al. (2010) Cancer Res. 70:1845-1853) or Gleevec (imatinib, Balachandran et al. (2011) Nat. Med. 17:1094-1100). In certain embodiments, the IDO1 pathway inhibitor (e.g., an inhibitor of a downstream signaling pathway) is 1-methyl-tryptophan, particularly 1-methyl-D-tryptophan (indoximod, NLG-8189, 1-methyl-D-tryptophan; NewLink Genetics), comprising salts and prodrugs (U.S. Patent Application Publication No. 20170022157), or a racemic mixture having the same.

[0017] Inhibitors of IDO1 expression include, but are not limited to, JAK / STAT (e.g., JAK, STAT3, STAT1) (Du et al. (2000) J. Interferon Cytokine Res., 20:133-142, Muller et al. (2005) Nature Med., 11:312-319; Yu et al. (2014) J. Immunol., 193:2574-2586), NFκB (Muller et al. (2005) Nature Med., 11:312-319; Muller et al. (2010) Cancer Res., 70:1845-1853), KIT (Balachandran et al. (2011) Nature Med., 17:1094-1100), and MET (Rutella et al. (2006) Blood This includes inhibitors of RAS / RAF / MEK (Liu (2010) Blood 115:3520-3530), aryl hydrocarbon receptor (AHR) (Bessede et al. (2014) Nature 511:184-190; Litzenburger et al. (2014) Oncotarget 5:1038-1051), or vascular endothelial growth factor receptor (VEGFR) (Marti et al. (2014) Mem Inst Oswaldo Cruz 109:70-79). In certain embodiments, the inhibitor is not a VEGFR inhibitor.

[0018] Inhibitors of the IDO1 downstream signaling pathway include, but are not limited to, GCN2 (Munn et al. (2005) Immunity 22:633-642; Muller (2008) Proc Nat Acad Sci., 105:17073-17078), C / EBP homologous protein 10 (CHOP-10, also known as gadd153, hereinafter referred to as CHOP; Munn et al. (2005) Immunity 22:633-642), activating transcription factor 4 (ATF4; Munn et al. (2005) Immunity 22:633-642; Thevenot et al. (2014) Immunity 41:389-401), or aryl hydrocarbon receptor (AHR) (Opitz et al. (2011) Nature 478:197-203; This includes inhibitors of Litzenburger et al. (2014) Oncotarget 5:1038-1051), or inhibitors of activators of mammalian targets of rapamycin (mTOR) or protein kinase C (PKC)-θ (Metz et al. (2012) Oncoimmunology 1:1460-1468). In certain embodiments, the inhibitor of the IDO1 downstream signaling pathway is an inhibitor of IL6 (e.g., an antibody immunologically specific to IL6).

[0019] In certain embodiments, the agent administered with the IDO1 signaling inhibitor is an anti-angiogenic agent and / or therapeutic agent that acts by imposing nutrient / oxygen starvation (e.g., starvation or hypoxia) or that utilizes nutrient / oxygen starvation in the target tissue. For example, the anti-angiogenic agent and / or therapeutic agent may be, but is not limited to, 1) an anti-angiogenic agent, 2) a hypoxia-activated prodrug or bioreducing agent, 3) a targeted agent that utilizes the response to hypoxia (e.g., HIF-targeted agents), or 4) a molecularly targeted agent that utilizes the response to stress due to nutrient or oxygen deficiency (antimetabolites, UPR inhibitors, etc.).

[0020] In certain embodiments, the anti-angiogenic agent is a chemotherapeutic agent having anti-angiogenic properties. In certain embodiments, the anti-angiogenic agent is a VEGF antagonist that can neutralize, block, inhibit, suppress, reduce or interfere with VEGF activity, including binding to one or more VEGF receptors. Examples of VEGF antagonists include, but are not limited to, anti-VEGF antibodies, VEGF traps, anti-VEGFR antibodies, VEGFR inhibitors, thalidomide, and DI. These may include 14-Notch inhibitors, antitubulin vasoconstrictors (VDAs), angiopoietin-Tie2 inhibitors, nitric oxide synthase (NOS) inhibitors, cationic polyamino acid dendrimers, rapamycin, everolimus, temserolimus, low molecular weight heparin, SPARC (osteonectin) peptides, bevacizumab, ranibizumab, ramucirumab, aflibercept, interleukin-17 (IL-17), DC101, sunitinib, sorafenib, pazopanib, AMG706, sediranib, vandetanib, vargatef, brivanib, XL-184, axitinib, tivozanib, thalidomide, lanalidomide, DMXAA, nadroparin, 2,5-dimethyl-celecoxib, cyclophosphamide, HBC, and tascinimod.

[0021] Examples of hypoxia-activated prodrugs / biodegrading agents include, but are not limited to, PR-104 (Proacta; La Jolla, CA, Patterson, et al. (2007) Clin. Cancer Res. 13:3922-3932), evophosphamide (TH-302, Duan, et al. (2008) J. Med. Chem. 51:2412-2420), tarloxotinib (TH-4000), apadiquan (EO9; Oostveen, et al. (1987) Tetrahedron 43:255-262), banoxantrone (AQ4N; Raleigh, et al. (1998) Int. J. Radiat. Oncol. Biol. Phys. 42:763-767), tirapazamine (TPZ; (Shinde, et al. al. (2009) J. Am. Chem. Soc. 131:14220-14221), SN30000 (CEN-209; Hicks, et al. (2010) Clin. Cancer Res. 16:4946-4957), CH-01 (Cazares-Korner, et al. (2013) ACS Chem. Biol. 8:1451-1459), BCCA621C (Lindquist, et al. (2013) Tumour Microenv. Ther. 1:46-55), O6-alkylguanine DNA alkyltransferase inhibitor (Zhu, et al. (2013) J. Med. Chem. 56:1355-1359), hypoxia-selective EGFR inhibitor (Karnthaler-Benbakka, et al. (2014) This includes Agnew Chem. Int. Ed. Engl. 53:12930-12935, or siRNA targeting hypoxia (Perche, et al. (2014) Agnew Chem. Int. Ed. Engl. 53:3362-3366).

[0022] In certain embodiments, the molecularly targeted drug that utilizes the response to hypoxia is an inhibitor of HIF-1α / HIF-1β activity (e.g., HIF-1α inhibitors). Examples of HIF activity inhibitors are provided by Wigerup et al. (Pharmacol. Ther. (2016) 164:152-169) (incorporated herein by reference). In certain embodiments, the HIF-1α inhibitor is an mRNA / protein expression inhibitor (e.g., PI3K inhibitors (e.g., Wartmannin, LY94002, GDC-0941, PI-103), mTOR inhibitors (e.g., rapamycin, PP242), aminoflavones, glyceoline, topotecan, PEG-SN38, EZN-2968, 2ME2, ENMD-1198, geldanamycin and analogs, bolus These include nostat (YC-1, PX-478, PX-12, pleurotine, cardiac glycosides, FM19G11, HIF-2α translation inhibitors), HIF-α / HIF-1β dimerization inhibitors (e.g., acriflavin, PT2385), DNA binding inhibitors (e.g., echinomycin, polyamides), and transcriptional activity inhibitors (e.g., ketomin, bortezomib, amphotericin B, tryptolide, AJM290, AW464).

[0023] In certain embodiments, molecularly targeted drugs that utilize responses to stress induced by nutrient or oxygen deficiency are antimetabolites. In certain embodiments, the antimetabolite is 2-mercaptopropionylglycine disulfide (TTL-315; DuHadaway et al. (2016) Oncotarget 7(7):7372-7380), a disulfide-containing compound that inhibits cell survival in a glucose-deficient manner. In certain embodiments, the agent is a disulfide-containing compound listed in U.S. Patent No. 20140079812, but is not limited thereto. For example, a disulfide-containing compound is a dialkyldisulfide (e.g., a lower alkyl disulfide containing at least one sulfur atom) or a diaryldisulfide, where the disulfide members may be the same (symmetric disulfide) or different (asymmetric disulfide). In other embodiments, the disulfide-containing compound is a thiamine-containing disulfide, for example, but not limited to, thiamine disulfide, thiamine propyl disulfide, and thiamine tetrahydrofuryl disulfide. In other embodiments, exemplary disulfide-containing compounds include, but not limited to, hydroxyethyl disulfide (HEDS; disulfide of mercaptoethanol (ME)), disulfide of mercaptopropionylglycine (MPG), disulfide of MPG and lower alkyl, disulfide of MPG and ME, disulfide of mesna (2-sulfanyethanesulfonate), disulfide of MPG and mesna, and disulfide of ME and mesna. In a particular embodiment, the disulfide-containing compound is the disulfide of MPG. In a particular embodiment, the disulfide-containing compound is HEDS.

[0024] In certain embodiments, molecularly targeted drugs that exploit responses to nutrient or oxygen deficiency stress are directed towards cellular stress signaling pathways. Examples of stress signaling pathways include, but are not limited to, the endoplasmic reticulum stress response (UPR) and antioxidant responses. An example of a drug that counteracts UPR is GSK2656157, a highly selective small molecule inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (Axten et al. (2013) ACS Med Chem Lett 4(10):964-968). PERK mediates the sequential activation of UPR by phosphorylating eukaryotic initiation factor 2 (eIF2), halting global cap-dependent translation while activating stress signaling aligned with the mitigation of physiological stress states (Tabas and David et al (2012) Nat Cell Biol 13(3): 184-190). PERK activation is widely observed in various tumors in response to severe hypoxia in their microenvironment and has been shown to be important for the effective survival and proliferation of tumor cells (Bi et al (2005) EMBO J 24 (19): 3470-3481). PERK inhibitors (GSK2656157 and GSK2606414) have been shown to have good oral bioavailability with low to moderate blood clearance in mice, rats, and dogs (Axten et al. (2013) ACS Med Chem Lett 4(10):964-968; Atkins et al (2013) Cancer Res. 73(6): 1993-2002; Axten et al (2012) J. Med. Chem. 55(16):7193-207). Treatment with GSK2656157 resulted in delayed growth of various human xenograft tumors in mice without significant weight loss and / or impact on pancreatic insulin production where PERK is highly expressed (Atkins et al (2013) Cancer Res. 73(6): 1993-2002). Examples of agents for antioxidant responses include, but are not limited to, ZnPPIX, PEG-ZnPPIX, and SMA-ZnPPIX.Hypoxia causes an imbalance of ROS in the tumor microenvironment, which in turn activates NRF2, a master regulator transcription factor that modulates genes involved in inducing antioxidant responses in tumor cells. While effective inhibitors have not yet been developed against NRF2, its main downstream target, heme oxygenase 1 (HO-1), has been successful as an inhibitory target. HO-1 is the rate-limiting antioxidant enzyme that breaks down heme into carbon monoxide (CO), biliverdin, and ferrous iron. Along with NRF2, HO-1 expression is regulated by hypoxia-activated PERK and HIF1a, and has been shown to be upregulated in various tumors such as renal cell carcinoma and prostate cancer, conferring resistance to radiotherapy and photodynamic therapy (Dey, et al. (2015) JCI 125(7):2592-608; Lee, et al. (1997) J. Biol. Chem., 272(9):5375-5381; Berberat, et al. (2005) Clin. Cancer Res., 11(10):3790-3798). Zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 enzyme activity, has been shown to be effective in reducing lymphoma, lung cancer, and sarcoma in mice (Jozkowicz, et al. (2007) Antiox Redox Sig 9(12):2099-2117). Improved, more soluble HO-1 inhibitors such as PEG-ZnPPIX and SMA-ZnPPIX have shown potential antitumor effects and fewer side effects in mice (Sahoo, et al. (2002) Bioconjug Chem 13(5):1031-1038).

[0025] In certain embodiments, cancers that can be treated using the compositions and methods of the present invention include, but are not limited to, prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, stomach cancer (gastric cancer), endometrial cancer, brain tumor, glioblastoma, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head and neck cancer, pharyngeal cancer, skin cancer, melanoma, basal cancer, mesothelioma, lymphoma, leukemia, esophageal cancer, breast cancer, rhabdomyosarcoma, sarcoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, and choriocarcinoma. In certain embodiments, cancer forms a tumor. In certain embodiments, cancer is lung cancer. In certain embodiments, cancer includes lung metastases. In certain embodiments, cancer includes infiltration of IDO-expressing hematopoietic cells in the tumor.

[0026] The present invention also includes compositions having 1) at least one IDO1 signaling inhibitor, and / or 2) at least one antivascularizing / anti-angiogenic agent and / or therapeutic agent that acts by imposing nutrient / oxygen starvation on a target tissue or by utilizing nutrient / oxygen starvation in a target tissue. The composition may further comprise at least one carrier (e.g., a pharmaceutically acceptable carrier). In certain embodiments, the composition comprises 1) at least one IDO1 signaling inhibitor, 2) at least one antivascularizing / anti-angiogenic agent and / or therapeutic agent, and 3) at least one carrier (e.g., a pharmaceutically acceptable carrier). The present invention also includes a kit having a first composition comprising at least one IDO1 signaling inhibitor, and a second composition comprising at least one antivascularizing / anti-angiogenic agent and / or therapeutic agent. The first and second compositions may further comprise at least one carrier (e.g., a pharmaceutically acceptable carrier). The carriers of the first and second compositions do not have to be the same.

[0027] The agents of the present invention (e.g., at least one IDO1 signaling inhibitor, and at least one anti-angiogenic agent / anti-angiogenic agent and / or therapeutic agent acting by imposing nutrient / oxygen starvation on target tissue or by utilizing nutrient / oxygen starvation in target tissue) are generally administered to patients as pharmaceutical formulations. As used herein, the term “patient” refers to human or animal subjects. These agents may be used therapeutically under the guidance of a physician for the treatment of cancer.

[0028] Pharmaceutical formulations containing the agent of the present invention can be conveniently formulated for administration in acceptable media such as water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oils, detergents, suspensions, or suitable mixtures thereof. The concentration of the agent in the selected media may vary, and the media may be selected based on the desired route of administration of the pharmaceutical formulation. Its use in the pharmaceutical formulation is intended unless conventional media or agents are incompatible with the agent to be administered.

[0029] The appropriate dosage and administration plan for the drug according to the present invention for a specific patient may be determined by a physician, taking into account the patient's age, sex, weight, general medical condition, and the specific condition and severity to which the drug is being administered. The physician may also consider the route of drug administration, the pharmaceutical carriers to which the drug can be combined, and the bioactivity of the drug.

[0030] The selection of an appropriate pharmaceutical formulation depends on the chosen method of administration. For example, the agent of the present invention may be administered by direct injection into any cancerous tissue or surrounding area. In this case, the pharmaceutical formulation comprises the agent dispersed in a medium compatible with cancerous tissue.

[0031] Drugs may also be administered intravenously into the bloodstream or parenterally by subcutaneous, intramuscular, or intraperitoneal injection. Pharmaceutical formulations for parenteral injection are known in the art. When parenteral injection is chosen as a method of administering antibodies, procedures must be taken to ensure that a sufficient amount of molecules reach the target cells to exert a biological effect. The lipophilicity of the drug or the pharmaceutical formulation to which they are delivered can be increased to allow molecules to reach their target sites more effectively.

[0032] A pharmaceutical composition containing the drug of the present invention as an active ingredient, tightly mixed with a pharmaceutical carrier, can be prepared according to conventional pharmaceutical formulation techniques. The carrier can take a wide variety of forms depending on the desired form of the formulation for administration. In the preparation of oral dosage forms, any of the usual pharmaceutical media can be used. For example, in the case of oral liquid formulations (e.g., suspensions, elixirs, and solutions), water, glycol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc., can be used. In the case of oral solid formulations (e.g., powders, capsules, and tablets), carriers such as starch, sugar, diluents, granulators, lubricants, binders, and disintegrants can be used. Due to their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms when a solid pharmaceutical carrier is clearly used. If necessary, tablets can be sugar-coated or enterically coated using standard methods. In the case of parenteral formulations, the carrier usually contains sterile water, but other components may be included, for example, to aid solubility or for preservation purposes. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspensions, etc., can be used.

[0033] The pharmaceutical formulations of the present invention may be formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically distinct unit of the pharmaceutical formulation suitable for the patient being treated. Each dosage should contain an amount of active ingredient calculated to produce the desired effect in relation to a selected pharmaceutical carrier. Procedures for determining an appropriate unit dosage form are well known to those skilled in the art. The unit dosage form can be increased or decreased proportionally based on the patient's body weight. An appropriate concentration for alleviating a particular pathological condition can be determined by dose-concentration curve calculation, as is known in the art.

[0034] In accordance with the present invention, the appropriate unit dosage form for administering the agent of the present invention can be determined by evaluating the toxicity of the agent in animal models. Various concentrations of the agent of the present invention can be administered to mice transplanted with human tumors, and the minimum and maximum doses can be determined based on the results of a significant reduction in tumor size and side effects as a result of the treatment. The appropriate unit dosage form can also be determined by evaluating the efficacy of the agent in combination with other standard anticancer agents. The unit dosage form of the agent can be determined individually or in combination with each anticancer treatment, according to the greater tumor contraction and / or reduction in growth rate.

[0035] The composition containing the agent of the present invention is administered at appropriate intervals, for example, at least twice a day, until pathological symptoms are reduced or alleviated, after which the dose may be reduced to a maintenance level. The appropriate interval in a particular case usually depends on the patient's condition.

[0036] definition The following definitions are provided to facilitate understanding of the present invention:

[0037] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise.

[0038] The “therapeutic effective dose” of a compound or pharmaceutical composition refers to the amount effective in preventing, inhibiting, treating, or alleviating the symptoms of a particular disorder or disease. For example, the “therapeutic effective dose” may refer to an amount sufficient to reduce the cancer burden in a subject.

[0039] "Pharmacologically acceptable" means that it has been approved by a federal or state regulatory agency, or has been approved for use in animals, particularly humans, as listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0040] "Carrier" refers to a vehicle administered together with, for example, a diluent, adjuvant, excipient, auxiliary agent, or activator of the present invention. Pharmaceutically acceptable carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or saline solution, as well as dextrose and glycerol solutions, are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are listed in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack Publishing Co., Easton, PA), Gennaro, AR, Remington: The Science and Practice of Pharmacy (Lippincott, Williams and Wilkins), Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0041] As used herein, the term “preventive” refers to preventive treatment of an individual at risk of developing a condition, resulting in a reduced probability of that individual developing the condition.

[0042] As used herein, the term “to treat” refers to any type of treatment that benefits a patient suffering from a disease, including, for example, improvement of the patient’s condition (e.g., one or more symptoms), or delaying the progression of the condition.

[0043] As used herein, the terms “host,” “subject,” and “patient” refer to any animal, including mammals such as humans.

[0044] As used herein, the term “small molecule” refers to a substance or compound having a relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic but are not proteins, polypeptides, or nucleic acids.

[0045] The term “small interfering RNA (siRNA)” refers to short (typically less than 30 nucleotides in length, particularly 12–30 or 20–25 nucleotides) double-stranded RNA molecules. siRNAs typically regulate the expression of genes targeted by the siRNA. Methods for identifying and synthesizing siRNA molecules are known in the art (see, for example, Ausubel et al. (2006) Current Protocols in Molecular Biology, John Wiley and Sons, Inc). As used herein, the term siRNA may include short hairpin RNA molecules (shRNA). Typically, shRNA molecules consist of short complementary sequences separated by a small loop sequence in which one of the sequences is complementary to the gene target. shRNA molecules are usually processed into siRNA in cells by an endonuclease. Exemplary modifications to siRNA molecules are provided in U.S. Patent Application Publication No. 20050032733. Expression vectors for the expression of siRNA molecules preferably use a strong promoter that can be constitutive or modulated. Such promoters are well known in the art and include, but are not limited to, the RNA polymerase II promoter, the T7 RNA polymerase promoter, and the RNA polymerase III promoters U6 and H1 (see, for example, Myslinski et al. (2001) Nucl. Acids Res., 29:2502 09).

[0046] An "antisense nucleic acid molecule" or "antisense oligonucleotide" contains a nucleic acid molecule (e.g., a single-stranded molecule) that targets (complementarily) a sequence (e.g., a translation initiation site and / or splice site) selected to inhibit the expression of a target protein. Such antisense molecules are typically about 15 to 50 nucleotides in length, more specifically about 15 to 30 nucleotides, and often extend to the translation initiation site of an mRNA molecule. Antisense constructs containing the entire sequence of the target nucleic acid molecule in the reverse direction can also be generated. Antisense oligonucleotides targeting any known nucleotide sequence can be prepared by oligonucleotide synthesis according to standard methods.

[0047] An “antibody” or “antibody molecule” is any immunoglobulin, including an antibody and its fragments, that binds to a specific antigen. As used herein, an antibody or antibody molecule intends to be a fusion of an intact immunoglobulin molecule, an immunoactive portion of an immunoglobulin molecule, and an immunoactive portion of an immunoglobulin molecule.

[0048] The antibody may be a naturally occurring antibody or a synthetic or modified antibody (e.g., recombinant antibody; chimeric antibody; bispecific antibody; humanized antibody; camelid antibody, etc.). The antibody may contain at least one purified tag. In certain embodiments, the framework antibody is an antibody fragment. The antibody fragment includes, but is not limited to, immunoglobulin fragments containing single domains (Dab; e.g., a single variable light or heavy chain domain) Fab, Fab', F(ab')2, and F(v), and, but is not limited to, fusions (e.g., via a linker) of these immunoglobulin fragments containing scFv, scFv2, scFv-Fc, minibodies, diabodies, triabodies, and tetrabodies. The antibody may also be a protein (e.g., a fusion protein) containing at least one antibody or antibody fragment.

[0049] The antibodies of the present invention may be further modified. For example, the antibodies may be humanized. In certain embodiments, the antibody (or a portion thereof) is inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and / or variable heavy domain of the antibody of the present invention may be inserted into another antibody construct. Methods for producing antibodies by recombination are well known in the art. In fact, commercially available vectors for specific antibodies and antibody fragment constructs are available.

[0050] The antibodies of the present invention may also be conjugated / linked to other components. For example, the antibody may be operably linked to at least one cell-permeable peptide, a detectable drug, a contrast agent, or a contrast agent (e.g., covalently linked via a linker as needed). The antibodies of the present invention may also comprise at least one purification tag (e.g., a His-tag). In certain embodiments, the antibody is conjugated to a cell-permeable peptide.

[0051] As used herein, the term “immunologically specific” refers to a protein / polypeptide, particularly an antibody, that binds to one or more epitopes of the protein or compound of interest but substantially does not recognize and bind to other molecules in a sample containing a mixed population of antigenic biomolecules.

[0052] The following examples are provided to illustrate various embodiments of the present invention. They are not intended to limit the invention in any way.

[0053] Examples Example 1 material and method Transgenic mouse strain BALB / c and C57BL / 6 strain background gene transfection Ido1 - / - Mice were obtained. BALB / c strain background gene transfection Ido2 - / - The development of the mouse is described (Metz et al. (2014) Int. Immunol., 26:357-367). Gene transfection in a BALB / c strain background.- / - and Il6 - / - The mouse strains, as well as the WT BALB / c and C57BL / 6 strains, were obtained from Jackson Laboratory (Bar Harbor, ME).

[0054] 4T1 tumor cell metastasis A study on lung metastasis using 4T1 mouse mammary cancer-derived cell lines (Aslakson et al. (1992) Cancer Res., 52:1399-1405) showed that 1 × 10⁶ cells were metastasized in 50 μl of serum-free medium. 4Orthotopic tumors were established by injecting cells into the fourth mammary fat pad, which then spontaneously metastasized to the lungs. To visualize the burden of lung metastases, lungs were inflated with 15% Indo ink dye, washed, and bleached with Fekete's solution. For immunofluorescence detection of the lung metastatic vascular system, lungs were inflated with 50% OCT, frozen in OCT blocks, and then 4 μm sections were prepared using the CryoJane tape transfer system. Vascular tissue within metastatic nodules was visualized by fluorescence staining with rabbit anti-mouse Caveolin1 polyclonal antibody (Cat. #3238S, Cell Signaling Technology, Boston, MA). To quantitatively assess vascular density within metastatic nodules, multiple fields were acquired per mouse lung using a Zeiss inverted microscope with a 40× objective lens. Pixel density corresponding to positive Cav1 signals per field was determined in Adobe Photoshop, and these values ​​were averaged to determine the overall average value per mouse. In the IDO1 inhibitor therapy study, mice in which metastasis was confirmed 3.5 weeks after 4T1 engraftment were force-administered 50 mg / kg of epacadostat (ChemieTek; Indianapolis, IN) in 100 μl of vehicle (3% N,N-dimethylacetamide, 10% 2-hydroxypropyl-β-cyclodextrin) twice daily over 72 hours. After 72 hours, the animals were euthanized for analysis. Positive control animals received a single intraperitoneal (ip) injection of 50 mg / kg of cyclophosphamide (Baxter; Deefield, IL) in 100 μl of sterile saline and were euthanized 72 hours later for analysis.

[0055] For immunofluorescence microscopy-based visualization of immune cells and IDO1-expressing cells in lung metastases, primary tumors and the spleen, resected tissue samples were frozen in OCT blocks, and then 4 µm sections were prepared using the CryoJane tape transfer system. For immune cells, according to the manufacturer's instructions, after staining with an antibody against the pan-immune cell surface marker CD45 conjugated to FITC (Cat. #103122, Biolegend), or antibodies against specific markers for characterizing MDSCs (myeloid-derived suppressor cells): FITC-conjugated Gr1 (Cat. #108419, Biolegend) and biotin-conjugated CD11b (Cat. #101204, Biolegend), followed by streptavidin-assembled Cy3 (Cat. #405215, Biolegend), immune cells were visualized by immunomicroscopy. The expression of IDO1 in cells was, as described in (Thomas et al. 20114 J Cell Biochem. 115:391-396), visualized by staining with IDO1-specific mouse monoclonal antibody 4B7 (Cat. #MABF850, Millipore), followed by staining with an FITC-conjugated goat anti-mouse secondary antibody (Cat. #F0257, Sigma) or a Cy3-conjugated goat anti-mouse secondary antibody (Cat. #M30010, Life Technologies). To isolate the IDO1-expressing immune cell population from 4T1 lung metastases, a single-cell suspension was prepared from resected metastatic lung tissue using a gentleMACS Octo Dissociator (Miltenyi Biotec) and a Tumor Dissociation Kit (Cat. #130-096-730, Miltenyi Biotec) following the manufacturer's instructions, and whole immune cells were isolated using αCD45-labeled magnetic beads (Cat. #130-052-301, Miltenyi Biotec) according to the manufacturer's instructions. IDO1-expressing Gr1 + CD11b -The cell population was isolated from the total immune cell population by FACS (fluorescence-activated cell sorting) using a FACSAria (Becton Dickinson) cell sorter. It exhibited a higher proportion of Gr1 cells, which are representative of conventional MDSCs (myelin-derived suppressor cells). + CD11b - The cell population was simultaneously isolated as a non-IDO1 expression control population. CD45 was used for sorting. + Cells were stained with αGr1 antibody conjugated to PerCP and αCD11b antibody conjugated to FITC. To evaluate the effectiveness of FACS-based cell isolation, Gr1 + CD11b - and Gr1 + CD11b + Cells from both cell populations were mounted on slides using Cytospin 3, stained with DAPI and IDO1-specific mouse monoclonal antibody 4B7, and then stained with Cy3-conjugated goat anti-mouse secondary antibody (Cat. #M30010, Life Technologies). To directly assess the ability of IDO1-expressing cells to induce angiogenesis in vivo, 2x10 cells in 250 μL PBS were used. 6 Selected cells were mixed with 250 μL of Matrigel on ice and subcutaneously injected into the back of recipient mice to form a Matrigel plug. Nine days post-transplant, the Matrigel plug was excised, photographed, and frozen as an OCT block for frozen sectioning. Blood vessels within the Matrigel plug were visualized by fluorescence staining using rabbit anti-mouse caveolin 1 polyclonal antibody (Cat. #3238S, Cell Signaling Technology). To quantify vascular density, multiple fields were acquired using a Zeiss inverted microscope and analyzed using Adobe Photoshop as described above.

[0056] statistical analysis Graphing and statistical analysis were performed using Prism 6 (GraphPad Software, Inc.). Bar graphs were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA using, where necessary, unpaired two-tailed Student's t-test, Tukey's multiple comparison test, or Dunnett's multiple comparison test. Significance of Kaplan-Meier survival curves was determined by a two-group log-rank test. The range of p-values ​​is as follows: ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05; ns, not significant.

[0057] result Ido1 was attacked by a 4T1 breast cancer tumor transplanted at the same site. - / - (Ido1 homozygous knockout) mice showed a significant delay in the development of lung metastases compared to WT (wild-type) controls (Figure 1A). - / - When metastases formed in the host, they were observed to have lower vascular density than those formed in wild-type animals (Figure 1B), and this significance was confirmed by quantitative analysis (Figure 1C; Δ=2.6-fold, P<0.01). This finding indicates that angiogenesis of 4T1 metastases in the lung is impaired in mice lacking IDO1.

[0058] Genetic data suggest that pharmacological inhibitors of IDO1 enzyme activity have therapeutic potential to reduce pathological angiogenesis. Epacadostat was administered by forced oral administration over 72 hours to mice with established 4T1 lung metastases. Epacadostat (INCB024360) is a specific small molecule inhibitor of IDO1 (Liu et al. (2010) Blood 115:3520-3530). Treatment with epacadostat resulted in a significant reduction in metastatic tumor angiogenesis compared to vehicle alone, comparable to the effect of the positive control compound cyclophosphamide (Δ=3.2-fold, P<0.01) (Ibe et al. (2001) J. Exp. Med., 194:1549-1559) (Figures 2A and 2B). These results confirm that blocking the enzymatic activity of IDO1 through pharmacological inhibition is sufficient to impair IDO1's ability to support angiogenesis.

[0059] The inflammatory cytokine IFNγ is a major inducer of IDO1. There is substantial evidence that IFNγ is important for effective antitumor immunity (Beatty et al. (2001) Immunol. Res., 24:201-210), and IFNγ-induced reduction in tumor neovascularization may be a mechanism of action (Qin et al. (2000) Immunity 12:677-686; Qin et al. (2003) Cancer Res., 63:4095-4100). Here, we investigated the effects of loss of both IFNγ and IDO1 on angiogenesis in 4T1 lung metastases in host animals. - / -Metastases formed in the host showed significantly lower vascular density compared to those formed in wild-type controls, while the loss of both IFNγ and IDO1 in double knockout mice completely counteracted the effect of IDO1 loss alone on 4T1 metastatic angiogenesis (Figures 3A and 3B). IDO1 loss resulted in a significant survival benefit for mice challenging orthotopic 4T1 tumors by reducing the lung metastatic burden (Smith et al. (2012) Cancer Discov., 2:722-735). In this context, simultaneous loss of IFNγ along with IDO1 in double knockout mice counteracted both the reduction in lung metastatic burden and the survival benefit observed with IDO1 loss alone (Figures 3C and 3D). These data demonstrate a surprising correspondence between the counteracting effects of IDO1 and IFNγ on angiogenesis and metastatic survival, suggesting that IDO1's antagonistic effect on IFNγ's anti-angiogenicity is directly linked to its pro-tumorogenic role in this metastasis model.

[0060] Loss of IDO1 is associated with reduced IL6 induction in 4T1 metastasis models, and metastatic susceptibility can be restored by providing exogenous IL6 (Smith et al. (2012) Cancer Discov., 2:722-735). Loss of IL6 affects angiogenesis in 4T1 lung metastases, and Il6 - / - Metastatic tumors obtained from animals showed significantly reduced vascular density compared to wild-type controls (Figures 4A and 4B). Again, the co-elimination of IFNγ suppressed the decrease in vascular density observed with the elimination of IL6 alone (Figures 4A and 4B). Similarly, IL6 loss was associated with a clear reduction in pulmonary metastatic burden and increased survival benefit in mice challenging orthotopic 4T1 tumors, benefits lost with the co-elimination of IFNγ (Figures 4C and 4D). The corresponding similarities in the effects of IL6 loss and IDO1 loss on susceptibility to angiogenesis and metastasis are consistent with IL6 functioning as a key downstream mediator of IDO's effects on these processes.

[0061] Elevated IDO1 enzyme activity is consistent with 4T1 metastatic proliferation in the lung (Smith et al. (2012) Cancer Discov., 2:722-735). To determine the types of cells expressing IDO1, the presence of IDO1 in tissue samples was detected by fluorescence microscopy using an IDO1-specific antibody. Immunofluorescence staining of metastatic pulmonary nodules revealed that IDO1 is expressed in tumor-infiltrating immune cells but not in tumor cells themselves (Figure 5A). + Immune cells were concentrated in the lung metastases but not in the primary 4T1 tumors or the spleen, suggesting that a specific IDO1 expression subtype is localized to 4T1 lung metastases or that IDO1 is specifically elevated in this particular microenvironment (Figure 5A). Further evaluation suggests that IDO1 in 4T1 lung metastases is elevated. + The cells were also found to be positive for the cell surface marker Gr1 (Figure 5B). Gr1 is one of the markers that define a mouse population of immune cells called MDSCs (myelin-derived suppressor cells). Another marker most commonly used to identify MDSCs is CD11b, but unexpectedly, IDO1 expression did not colocalize with this marker. Therefore, IDO1 is expressed in a population of immune cells that is associated with classical MDSCs but is different from them. Gr1 has been studied extensively for its immunosuppressive activity. + CD11b + Unlike MDSCs, Gr1 + CD11b - Little information has been reported about these cell populations, and there has been no previous association between these cells and IDO1 expression.

[0062] Gr1 expressing IDO1 supports angiogenesis. + CD11b - To functionally evaluate the role of the cell population, these cells were isolated from 4T1 lung metastases. Total CD45 + Immune cells were classified using flow cytometry, and Gr1 + CD11b - A subset was separated (Figure 6A). Gr1 represents the much more prevalent MDSC. + CD11b +The cell populations were simultaneously isolated as a control group of cells that do not express IDO1. Fluorescence microscopy analysis of these classified cell populations revealed that IDO1 positivity was Gr1. + CD11b - Limited to cells, CD11b positivity is Gr1 + CD11b + It was confirmed that the cells were limited to specific cells (Figure 6B). To evaluate the ability of a specific cell population to promote angiogenesis in vivo, FACS-isolated cells mixed with Matrigel were implanted subcutaneously to form subcutaneous plugs. After 9 days, the excised plugs were evaluated for invasive angiogenesis by both visual inspection and immunofluorescence staining of serial sections. Gr1 + CD11b - Cell-prepared plugs showed significant evidence of angiogenesis compared to negative control plugs without added cells (Figure 6C). Quantitatively, this is Gr1 + CD11b - The presence of the cells resulted in a 190-fold increase in vascular density (Figure 6D), confirming that this isolated cell population is sufficient to promote angiogenesis. In contrast, Gr1 + CD11b + In cell-prepared plugs, only minimal evidence of angiogenesis was observed (Figure 6C, 6D), and this activity was not indiscriminately associated with immature bone marrow cells, but rather with CD11b - This indicates subtype specificity. Gr1 + CD11b + To evaluate whether IDO1 expression in cells contributes to their ability to promote angiogenesis, FACS-isolated cells were subjected to Ido1 - / - Prepared from 4T1 lung metastases established in mice. In contrast to cells obtained from WT mice, Ido1 - / - Gr1 + CD11b - The cell is Gr1 + CD11b + Similar to the control group, these cells were ineffective in promoting angiogenesis (Figure 6C, 6D), indicating that IDO1 is essential for these cells' ability to promote angiogenesis.

[0063] The data provided herein establish a distinct biological role of IDO1 in supporting pathological angiogenesis and demonstrate that IDO1 facilitates this outcome through integration at a regulatory interface between two competing inflammatory cytokines, IFNγ and IL6. Angiogenesis was significantly reduced with loss of IDO1, and this effect was completely reversed with co-loss of IFNγ, a major inducer of IDO1. Loss of IL6, known to exhibit IDO1-dependent expression, similarly resulted in a decrease in angiogenesis that was determined to be IFNγ-dependent. Direct tumor relevance was confirmed in an orthotopic 4T1 lung metastasis model, in which corresponding effects on angiogenesis, metastatic tumor burden, and survival were observed.

[0064] While the tolerogenic properties of IDO1 have been extensively investigated, the potential influence of IDO1 on angiogenesis is not currently generally recognized, and a clear biological link has not been established. In particular, human xenograft tumors designed to overexpress exogenous IDO1 show increased vascular density (Nonaka et al. (2011) Int. J. Oncol., 38:113-120; Li et al. (2006) J. Invest. Dermatol., 126:128-136). Normal pulmonary angiogenesis is also reduced in IDO1 homozygous-deficient mice (Smith et al. (2012) Cancer Discov., 2:722-735). Current data suggest the importance of endogenous IDO1 in supporting angiogenesis and demonstrate a clear correlation between IDO1-dependent angiogenesis and the development of lung metastases. Current data also provide mechanistic evidence to explain the basis of this effect by linking IDO1 to regulatory interactions with inflammatory cytokines IFNγ and IL6.

[0065] In addition to expression in immune cells such as dendritic cells and macrophages, IDO1 expression has also been confirmed in endothelial cells (Blaschitz, et al. (2011) PLoS One 6:e21774). Biochemically, IDO1 can signal via two different metabolic pathways by catabolizing tryptophan: one in response to downstream tryptophan catabolic metabolites and the other in response to the depletion of tryptophan itself. Both mechanisms are associated with the positive regulation of IL6 via kynurenine signaling mediated by AHR (aryl hydrocarbon receptor) or amino acid depletion signaling mediated by GCN2 (general nonderepressible 2) (Dinatale et al. (2010) Toxicol. Sci., 115:89-97; Liu et al. (2014) Mol. Cell. Biol., 34:428-438).

[0066] In addition to establishing the role of IDO1 in supporting angiogenesis, the data provided herein demonstrate that the use of pharmacological inhibitors is an effective intervention strategy for angiogenesis. In the 4T1 metastasis model, administration of epacadostat to mice harboring established angiogenic metastases resulted in a reduction in the level of angiogenesis in response to treatment. Therefore, this result indicates that treatment with IDO1 inhibitors effectively reduces established angiogenic networks, rather than simply repeating the role of IDO1 in supporting angiogenic development. The antivascular effect of cyclophosphamide is IFNγ-dependent (Ibe et al. (2001) J. Exp. Med., 194:1549-1559), and if not directly linked, mechanical convergence by IDO1 has also been suggested.

[0067] IDO1 small molecule inhibitors are currently being evaluated in various cancer clinical trials based on the assumption that they help enable an effective immune response against tumors. However, this research has shown that angiogenesis is also involved. The established clear interrelationship between the effects of IDO1, IFNγ, and IL6 loss on angiogenesis and metastatic survival is consistent with other indicators of the importance of IFNγ's anti-angiogenic effect on tumors (Qin et al. (2000) Immunity 12:677-686; Qin et al. (2003) Cancer Res., 63:4095-4100). Therefore, when evaluating the clinical outcomes obtained with IDO1 inhibitors, the impact on the tumor vascular system should be further considered, especially in the context of lung metastases.

[0068] An emerging concept in the field of angiogenesis is that normalizing the vascular structure of pathological tumors enhances the effectiveness of chemotherapy and radiotherapy. Synergistic responses obtained by combining IDO1 signaling inhibitors and chemotherapeutic agents have been observed in different mouse tumor models (Hou et al. (2007) Cancer Res., 67:792-801; Muller et al. (2005) Nat. Med., 11:312-319). Furthermore, clinical trials have indicated that indoximod, an IDO1 pathway inhibitor, appears to sensitize tumors to salvage chemotherapy (Soliman et al. (2013) J. Clin. Oncol., 31(suppl): abstr 3069), which is consistent with the normalization of blood vessels that improves the response to chemotherapy. IDO1 inhibition may also enhance IFNγ-dependent anti-angiogenic effects induced by certain chemotherapeutic agents such as cyclophosphamide, which has been reported to cause immune-dependent rejection of large angiogenic tumors mediated by the destruction of tumor angiography in a manner that requires IFNγ receptor expression in normal host cells (Ibe et al. (2001) J. Exp. Med. 194:1549-1559). By eliminating the negative feedback constraints imposed on the anti-angiogenic effects of IFNγ by IDO1, IDO1 inhibitors can enhance this aspect of the therapeutic response to other chemotherapeutic agents identified as having anti-angiogenic properties, such as cyclophosphamide and taxanes (Bocci et al. (2002) Cancer Res., 62:6938-6943).

[0069] Example 2 material and method In a study investigating tumor hypoxia and treatment with IDO1 inhibitors, mice with established metastasis 3.5 weeks after 4T1 engraftment were orally administered either 50 mg / kg of epacadostat (ChemieTek; Indianapolis, IN) in 100 μl vehicle (3% N,N-dimethylacetamide, 10% 2-hydroxypropyl-β-cyclodextrin) twice daily over 72 hours, or 400 mg / kg of indoximod (Sigma-Aldrich; St. Louis, MO) in 100 μl vehicle (0.5% Tween 80 / 0.5% methylcellulose v / v aqueous solution) twice daily over 72 hours.

[0070] To visualize hypoxic areas within 4T1 lung metastases, Hypoxyprobe TM Green Kit (Hydroxyprobe, Inc.; Burlington, MA) was used according to the manufacturer's instructions. One hour before euthanasia, mice were given 60 mg / kg of Hypoxyprobe. TM Pimonidazole hydrochloride was administered intravenously. Lung sections were stained with a supplier-provided FITC-labeled mouse IgG1 monoclonal antibody (4.3.11.3) that specifically binds to the protein adduct of pimonidazole.

[0071] result As described above, administration of an IDO1 inhibitor reduced angiogenesis in metastatic tumors (Figure 2). Here, it was determined whether a corresponding increase in hypoxia due to insufficient blood supply to the tumor occurred. Specifically, the IDO1 inhibitor epacadostat was force-administered orally to mice with established 4T1 lung metastases over 72 hours. Before euthanasia, the mice in this experiment were euthanized using Hypoxyprobe, a reagent that preferentially labels hypoxic areas in tissues. TM He also received an injection of [substance name]. Immunofluorescence confocal microscopy images are shown in Figure 7. Treatment with epacadostat resulted in a significant reduction in angiogenesis in metastatic tumors compared to vehicle alone (Figure 7A). Similarly, Hypoxyprobe in metastatic tumors after epacadostat treatment. TMStaining showed clear evidence of hypoxia compared to vehicle-treated controls, as evidenced by the green staining areas present throughout the sections (Figure 7B). Similar results were obtained in 4T1 tumor-bearing mice treated with the IDO1 pathway inhibitor indoximod (Figures 8A and 8B). These results indicate the utilization of IDO1 inhibitors to inhibit angiogenesis by depriving tumors of nutrients and oxygen, thereby increasing sensitivity to drugs that target or exploit oxygen / nutrient starvation in cancer cells.

[0072] To provide a more complete description of the latest technologies relating to the present invention, several publications and patent documents are referenced in the aforementioned specification. The disclosures of each of these references are incorporated herein by reference.

[0073] While some preferred embodiments of the present invention have been described and specifically illustrated above, the invention is not intended to be limited to such embodiments. Various modifications can be made without departing from the scope and spirit of the invention, as described in the claims.

Claims

1. A composition for inhibiting angiogenesis in a subject having a tumor that has metastasized to the lung, wherein the composition is An inhibitor of at least one indoleamine 2,3-dioxygenase-1 (IDO1), The IDO1 inhibitor is 1-methyl-tryptophan, 1-methyl-D-tryptophan (indoximod), or a racemic mixture containing 1-methyl-D-tryptophan, epacadosat or napoxymod, an antibody that binds to and inhibits IDO1, or a nucleic acid molecule that inhibits IDO1 expression. The nucleic acid molecule that inhibits IDO1 expression is siRNA, shRNA, or antisense oligonucleotide. IDO1 inhibitors and At least one PERK inhibitor, which is GSK2656157 or GSK2606414, or at least one hypoxia-activated prodrug or bioreducing agent, which is PR-104, evophosphamide, or tarloxotinib, At least one pharmaceutically acceptable carrier and It has, In the above composition, the IDO1 inhibitor induces a tumor hypoxic state by inhibiting IDO1, thereby increasing the sensitivity of tumor cells metastasized to the hypoxic lung to the PERK inhibitor or the hypoxia-activating prodrug. composition.

2. The composition according to claim 1, wherein the IDO1 inhibitor is 1-methyltryptophan.

3. The composition according to claim 1, wherein the PERK inhibitor is GSK2656157.

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